WESTPAC workshop on the development of a research strategy for Harmful Algal Blooms “Toward the future development of HAB science in the Western Pacific –What we know, and what we do not know on HABs” National Research Institute of Fisheries Science T oshiyuki Suzuki • Toxins (DSP, AZAs, BTXs, etc.) and toxic species • Chemical nature, symptoms, action mechanism, and analysis of toxins • What we know, and what we should do for toxins on the HAB
Marine Biotoxins • Shellfish toxins + Paralytic shellfish toxins + Diarrhetic shellfish toxins + Neurotoxic shellfish toxins (Brevetoxins) + Amnesic shellfish toxins (domoic acid) + Azaspiracids • Fish toxins + Puffer fish toxins (tetrodotoxins) + Ciguatera fish toxins (ciguatoxins) etc +.others (palytoxin and ovatoxins) • Others + Cyclic imines etc
Paralytic shellfish Diarrhetic shellfish poisoning T oxic poisoning dinoflagellates and H 2 N O structure of toxins 1 3 O H H NH N 7 + 1 Okadaic acids NH 2 8 Dinophysis fortii + 9 NH H 2 N Me N OH 1 OH O 2 1 OH O O Me HO 1 Alexandrium O O O OH Me Saxitoxins tamarense Me O O OH OH Me Bivalves shellfish Amnesic Several shellfish poisoning CH 3 toxins Clip CH 2 COOH Pseudonitzchia multiseries COOH N H HOOC CH 3 ドーモイ酸 H Neurotoxic shellfish Mussels Scallops poisoning Azaspiracid poisoning O H O HO H O H O Gymnodinium O H Me OH mikimotoi NH 2 HO O Me Azaspiracids S O COOH Me OH H H Me Me OH O H H O Me H NH H Me O Me O Me O O H H O O O H H O O H Protoperidinium O O O H H H H H O Me H H crassipes Me Brevetoxins Me Me
Diarrhetic Shellfish Poisoning (DSP) and other lipophilic toxins Dinophysis spp. OH O O R1 O HO O O O O OH O OR 3 R2 OH
Dinophysis spp. OH O O R1 O HO O O O O OH O OR 3 R2 OH R1 R2 R3 CH 3 Okadaic acid H H Diarrhetic Shellfish Poisoning CH 3 CH 3 Dinophysistoxin-1 H CH 3 CH 3 acyl (palmitoyl) Dinophysistoxin-3 T oxins (OA, DTXs) Symptoms: diarrhoea, nausea, vomiting, abdominal pain O Me Me O O O O O R1 C7 O OH OH OH CH 2 OH Pectenotoxin-1 R Me O Me O Pectenotoxin-2 CH 3 R R O Pectenotoxin-3 CHO R O Me Me Pectenotoxin-6 COOH R Pectenotoxins Symptoms: ?
Ｄｉｎｏｐｈｙｓｉｓ species (by Prof. Y . Fukuyo,The University of T okyo)
Protoceratium reticulatum OH Me R1 O R1 Y essotoxin H 45-Hydroxy-yessotoxin OH O O Me OH O O Me O NaO SO O O 3 Y essotoxins Me O Symptoms: ? Me NaO 3 SO O O
Chemistry of DSP and other lipophilic toxins Lipophilic, soluble in MeOH etc. Chemical conversion Alkaline hydrolysis of OA and DTX esters • Acid catalyzed interconversion of PTXs • Biological conversion Enzymatic acylation of OA and DTX1 in shellfish • Enzymatic hydrolysis and conversions of PTXs in • shellfish
OH O O R2 1 O R 4 O O O O O OH O 7 OR 1 R3 OH R1 R2 R3 R4 CH 3 okadaic acid (OA) H H H free toxins dinophysistoxin-1 (DTX1) H CH 3 H CH 3 dinophysistoxin-2 (DTX2) H H CH 3 H dinophysistoxin-3 (DTX3) acyl CH 3 CH H 7- O -acyl-esters 3 OA diol esters R1 R2 R3 R4 1-diol-esters OA D8 H CH3 H a DTX4 H CH3 H b H 2 a C OH O OSO 3 H H 2 b OH C OH OH O OSO 3 H OSO 3 H
Enzymatic acylation of OA and DTX1 in shellfish CH 3 OH O CH 3 CH 2 O O O HO O O O O CH 3 OH OH CH 3 CH 3 OH CH 3 + OH O Shellfish enzyme CH 3 OH O CH 2 CH 3 O O O HO O O O O CH 3 OH CH 3 OH CH 3 CH 3 O O
Alkaline hydrolysis of OA and DTX esters OA esters (OA diolesters or 7- O acyl-OA) CH 3 OH O R 2 O CH 2 O 2 31 O R 2 O O O O 7 O CH 3 OH 35 OCOR 1 CH 3 R 3 OH CH 3 0.5M NaOH/ aqueous MeOH 75C, 40 min Free OA CH 3 OH O CH 2 R 2 O O 31 2 O HO O O O 7 O CH 3 OH 35 OH CH 3 R 3 OH CH 3 + O R1COOH C16:0 HO R2OH
Toxicology of DSP and other lipophilic toxins MBA MBA by Characteristic by i.p. oral toxicities OA/ DTXs diarrhea, tumor promoting ○ ○ PP2A inhibition PTXs (No actin polymerization inhibition ○ potent cytotoxicity human Severe damage to liver by i.p intoxication) YTXs (No E-cadherin fragmentation ○ Modulate the Ca 2+ homeostasis human intoxication)
Regulatory level of DSP and other lipophilic toxins Japan EU CODEX （ mg/kg ） (mg/kg) (mg/kg) OA, DTXs 0.16 0.16 0.16 (DSP toxins) YTXs - 3.7 - PTXs - 0.16 -
PP2A inhibition assay kit for DSP toxins
Principle of PP2A inhibition assay OAs PP2A OH O O R1 O HO O O O MW(Active subunit) O O OH OR 3 ： OH R2 35 kDa R1 R2 R3 CH 3 H Okadaic acid H Binding CH 3 Dinophysistoxin-1 CH 3 H CH 3 acyl (palmitoyl) Dinophysistoxin-3 CH 3 O OH P OH O O 2 N Hydrolysis Inactivation of PP2A O OH P OH HO O O O 2 N O 2 N OH P OH pNPP : p-Nitrophenyl Phosphate H × pNP : p-Nitrophenol HO ydrolys O OH P OH O O 2 N is
LC-MS vs protein phosphatase 2A inhibition assay *1 for okadaic acid analogues 20.00 y = 1.2448x + 0.1165 R 2 = 0.9744 16.00 free + esterified OA analogues PP2A (mg/kg) 12.00 8.00 y = 0.9622x + 0.0167 4.00 R 2 = 0.9905 free OAanalogues 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 LC-MS (mg/kg)
Japanese ongoing research project for producing reference toxins by combining algal culture and chemical and enzymatic reactions OA, DTX1 PTX2 Enzymatic conversions Dinophysis spp. PTX1,3,6 YTX OA, DTX1 Protoceratium reticulatum Prorocentrum lima National Research Institute of Fisheries Science Food Hygiene & Development research group
What we know • DSP is characterized by gastrointestinal symptoms, including: nausea, vomiting, diarrhea, abdominal pain, headache, and fever. Symptoms develop from 30 minutes to 3 hours after consumption and last for up to 4 days. • Dinophysis spp and Prorocentrum spp are the causative species producing okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2) and OA diol esters. • Dinophysistoxin-3 (DTX3) includes a wide range of derivatives of OA, DTX1, and DTX2, esterified with saturated and unsaturated fatty acids, products of metabolic transformations that occur in the shellfish. • There are a few reports in which the presence of OA in bivalves has been associated with epibenthic dinoflagellates of the genus Prorocentrum spp. • Pectenotoxins and yessotoxins are eliminated from the regulation for marine toxins on the CODEX standard.
What we know • LC/MS/MS is an internationally accepted analytical method for testing of OA and DTXs. • PP2A inhibition assay is useful method for testing OA and DTXs although there are few commercially available testing kits. There are commercially available certified reference materials of OA • and DTXs. • There have been no documented occurrences of illness to date in the Southeast Asian region, however reports of this illness can be misidentified as a bacterial or viral source and is expected to be highly under-reported.
What we should do • Monitoring of Dinophysis spp in Southeast Asian region. • Investigation on the cellular toxin profiles and contents of Dinophysis spp. Chemical analyses of OA and DTXs in bivalves to assess the risk of • DSP in Southeast Asian region. Establish the monitoring system for DSP by LC/MS/MS or PP2A • inhibition assay for protection of humane health.
Neurotoxic Shellfish Poisoning (NSP) (Brevetoxins) Karenia breve HO H O O H O H H O H O H H O H O H H O O O O H H H H H H O Brevetoxin A
Neurotoxic Shellfish Poisoning T oxins (Brevetoxins) HO R Karenia brevis O O H H H H O O H HO H O H H O H O H O O H O O O H H H O O O H H O H H H H O H H O H O H H O O O O Brevetoxin B R= H H H H H CHO H O H Brevetoxin A Brevetoxin B1 R= N SO Na 3 O NH 2 CH 2 OH Brevetoxin B2 R= S COOH O Lipophilic, soluble in MeOH etc. Symptom: nausea, vomiting, diarrhoea, chills, sweats, reversal of temperature, hypotension, paresthesias of lips, paralysis
Neurotoxic Shellfish Poisoning T oxins (Brevetoxins) A few poisoning cases have been reported from USA and New Zealand. Florida, T exan, North Carolina (USA) Human poisoning cases by consumption of bivalves in the bloom of toxic dinoflagellate Karenia brevis ( Gymnodnium breve ) (1962). New Zealand cases by consumption of Greenshell Human poisoning mussels (1992). BTXs are originally reported as ichthyotoxic compound.
Working mechanism of Brevetoxins Opening of the voltage-gated sodium channel outside Na channel Paralytic shellfish toxins Na + =145 mM Tetrodotoxins Na + Na + K + =5 mM Ciguatoxins U U U U U U U U U U U Receptor-1 Brevetoxins U U U U U U U U U U U Channel inside close Na + =10 mM K + K + =140 mM Receptor-5 U U U U U U U U U U U U Channel U U U U U U U U U U U U open outside Voltage-gated sodium channel cell membrane
Internationally approved maximum level/kg of marine biotoxins in sea food Biotoxins Poisoning Maximum level Saxitoxin group PSP 0.8 mg Okadaic acid group DSP 0.16 mg Domic acid group ASP 20 mg Brevetoxin group NSP 200 MU Azaspiracid group AZP 0.16 mg
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